Affinity chromatography of vibrio cholerae enterotoxin-ganglioside polysaccharide and the biological effects of ganglioside-containing soluble polymers

ABSTRACT

Columns of polysaccharide, e.g., agarose, derivatives comprising covalently bonded gangliosides quantitatively adsorb the  125  I-labeled cholera toxin of chromatographed samples. The most preferred derivatives are those wherein the gangliosides are coupled to &#34;macromolecules&#34; [native albumin, denatured albumin, poly(L-lysine) and poly(L-lysyl-DL-alanine) graft copolymers] which themselves are covalently bonded to the, e.g., agarose. The soluble ganglioside polymers can prevent the binding of  125  I-labeled toxin to liver membranes as well as block completely the lipolytic activity of cholera toxin on fat cells, and thus are useful in the management of the manifestations of clinical cholera.

This is a continuation, of application Ser. No. 475,313, filed May 31,1974, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the preparation and use of certaininsoluble polysaccharide derivatives comprising covalently linkedgangliosides, and, more especially, relates to the use of suchderivatives for the extraction and purification of cholera toxin byaffinity chromatography techniques.

2. Cross Reference to Related Applications

1. Serial No. 475,305, filed May 31, 1974, now U.S. Pat. No. 3,947,352;hereby expressly incorporated by reference and relied upon.

2. Serial No. 475,314, filed May 31, 1974, now abandoned, and copendingcontinuing application, Ser. No. 713,108, filed Aug. 10, 1976; herebyexpressly incorporated by reference and relied upon.

3. Description of the Prior Art

It has recently been demonstrated that the enterotoxin from Vibriocholerae, which is responsible for the gastrointestinal manifestationsof clincial cholera, binds very strongly to gangliosides and lessstrongly to certain glycoproteins such as fetuin and thyroglobulin.Cuatrecasas, Biochemistry, 12, 3547 (1973a); Cuatrecasas, Biochemistry,12, 3558 (1973b). Gangliosides block the biological effects of choleratoxin on isolated fat cells [Cuatrecasas, 1973a, supra; Cuatrecasas,1973b, supra; van Heyningen et al, J. Infec. Dis., 124, 415 (1971)] andon the small intestine [van Heyningen et al, supra; Pierce, J. Exp.Med., 137, 1009 (1973); Holmgren et al, Scand. J. Infec. Dis., 5, 77(1973)], and they prevent the binding of ¹²⁵ I-labeled cholera toxin tospecific receptors on the cell membranes of various tissues, such asadipose tissue, liver erythrocytes, and intestinal epithelial cells;Cuatrecasas, 1973a, b, supra. There is considerable evidence indicatingthat gangliosides, and specifically G_(M1) gangliosides, are the naturalmembrane receptors with which cholera toxin specifically interacts toelicit its biological effects in tissues; Cuatrecasas, 1973a, b, supra.

SUMMARY OF THE INVENTION

It has now been determined according to the invention that certaininsoluble polysaccharide, e.g., agarose, derivatives comprisingcovalently linked gangliosides are useful for the extraction andpurification of cholera toxin by affinity chromatography [Cuatrecasas etal, Proc. Nat. Acad. Sci. U.S., 61, 636 (1968); Cuatrecasas, Advan.Enzymol., 35, 29 (1972a); Cuatrecasas, Proc. Nat. Acad. Sci. U.S., 69,1277 (1972b)]. The utility of such insoluble biospecific adsorbents, aswell as of soluble polymers which contain covalently coupledgangliosides, in the therapeutic approach to clinical cholera isdemonstrated by evidencing that such derivatives effectively block thebinding and the metabolic effects of cholera toxin in isolatedadipocytes.

Briefly, according to the invention, columns of agarose derivatives[other polysaccharide derivatives include cellulose, starch, and thecross-linked polysaccharide gels, Sephadex and Sepharose] containingcovalently attached gangliosides quantitatively adsorb the ¹²⁵ I-labeledcholera toxin of chromatographed samples. The most effective derivativesare those in which the gangliosides are coupled to "macromolecules"[native albumin, denatured albumin, poly-L-lysine andpoly(L-lysyl-DL-alanine) graft copolymers] which are covalently linkedto agarose. Ganglioside adsorbents [1-ml columns] comprising suchmacromolecular "arms" can effectively adsorb cholera toxin even afterthe adsorbent is diluted 200- to 600-fold with unsubstituted agarose.Compare application, Serial No. 475,314, now abandoned, and copendingcontinuation application, Ser. No. 713,108, supra. Selective adsorptionis blocked if the toxin is incubated with free gangliosides beforechromatography. Quantitative elution is achieved with buffers containing5-7 M guanidine.HCl. The biological activity of purified samples ofcholera toxin is completely removed by chromatography on smallganglioside-agarose affinity columns, and this activity can bequantitatively recovered upon elution with guanidine.HCl. Small [5-ml]affinity columns can remove virtually all [more than 99%] of the choleratoxin activity and ¹²⁵ I-labeled toxin present as a tracer inpreparations of filtrates of Vibrio cholerae derived from about 41. ofcrude culture medium. Ganglioside-agarose beads can block the lipolyticeffects of cholera toxin on isolated fat cells. Soluble gangliosidepolymers, prepared by covalently attaching the glycolipids to branchedcopolymers of lysine and alanine (see again application, Serial No.475,314, now abandoned, and copending continuation application, Ser. No.713,108, supra), can prevent the binding of ¹²⁵ I-labeled toxin to livermembranes, as well as block completely the lipolytic activity of choleratoxin on fat cells. These polymeric ganglioside derivatives thus areuseful in the management of the manifestations of clinical cholera.Studies with sodium dodecyl sulfate disc gel electrophoresis indicate,albeit applicant does not wish to be bound by this theory, that choleratoxin is composed of two major subunits having molecular weights ofabout 66,000 and 36,000. Reduction and alkylation convert the largersubunit into components having a molecular weight of about 8,000, andthe smaller subunit is converted to two components having molecularweights of about 27,000 and 8,000; the mole of disulfide bonds inmaintaining or stabilizing the oligomeric structure of the two majorsubunits is uncertain. The larger subunit [molecular weight 66,000]appears to be very similar to or identical with choleragenoid, a toxinderivative which is antigenically very similar to toxin, which isbiologically inactive, and which competitively inhibits the binding andbiological activity of cholera toxin. The smaller subunit [molecularweight 36,000] does not appear to bind to cells. Hence, it is proposedthat the ability of cholera toxin to bind specifically to cells isgoverned solely by the larger subunit, but that the ability to elicit aspecific biological response resides in the smaller subunit. Choleratoxin is thus suggested to consist of one subunit which acts to deliverto the cell membrane, in a highly specific manner, another molecule[subunit] which in turn is capable of inducing subsequent changes whichlead to the biological response.

DETAILED DESCRIPTION OF THE INVENTION

General Properties of Affinity Adsorbents. Whereas [¹²⁵ I]-cholera toxindoes not bind to columns containing unsubstituted agarose, a substantialportion of the radioactivity does adsorb to columns containingfetuin-agarose or A-DADA-gang¹ ; compare FIG. 1, Table I and themethodology appurtenant thereto, Cuatrecasas et al, Biochemistry, 12,No. 21, 4253 at page 4255 (1973e), hereby expressly incorporated byreference and relied upon. About 80% of the radioactivity in theiodinated toxin preparation can bind specifically to liver membranesbefore chromatography. About 30% of the radioactive material applied toa fetuin-agarose column is not adsorbed, and about 20% of this materialcan still bind selectively to liver membranes. The ganglioside-agaroseadsorbent appears to be more effective than that which contains fetuinsince 15-20% of the radioactive material which is applied to the columnappears in the breakthrough of the column, and virtually none of thismaterial can bind selectively to liver membranes. It appears that about15-20% of the total radioactive content of [¹²⁵ I]cholera toxinrepresents radioactivity on denatured or contaminating protein. Thespecificity of the adsorptive process is further illustrated bydemonstrating that incubation of the [¹²⁵ I]cholera toxin withgangliosides before chromatography effectively prevents the subsequentadsorption of radioactivity to the column.

In the experiments described in said FIG. 1, Cuatrecasas et al, 1973e,supra, elution of the adsorbed [¹²⁵ I]-toxin was achieved with 7 Mguanidine.HCl. Experiments were performed to determine whether milderconditions could be utilized to elute the toxin from such columns. Thestrength with which the toxin is adsorbed is evident from the inabilityto achieve elution with 0.1 M acetic acid, 2 N NaCl, and 3 Mguanidine.HCl containing 1 N NaCl. Even 4 M guanidine.HCl results in theelution of only about one-third of the bound toxin. Nearly quantitativeelution, however, can be obtained with higher concentrations (5 M) ofguanidine.HCl or with 0.1 N HCl.

Samples which had been chromatographed on A-DADA-gang columns such asthat described in the aforesaid Cuatrecasas et al, 1973e, FIG. 1contained detectable amounts of free gangliosides. Because the presenceof this compound in the samples can interfere with assays of thebreakthrough materials, and it can also potentially interfere withadsorption of the toxin to the column, experiments were performed todetermine if other derivatives were less susceptible to this "leakage"phenomenon [Table II, Cuatrecasas et al, 1973e, supra, at page 4256].Since the presence of free gangliosides in the column breakthroughsamples is meaningful only when considered in relation to theconcentration and effectiveness of the selective adsorbent, the variousderivatives were diluted serially with unsubstituted agarose and theirability to extract [¹²⁵ I]cholera toxin was compared. As predicted fromthe considerations described earlier, it is clear that the leakage offree gangliosides from the adsorbents which contain macromolecularspacers [native albumin, denatured albumin, poly-L-lysine andpoly(-L-lysyl-DL-alanine) graft copolymer] is much less marked than thatwhich occurs with A-DADA-gang. Of equal importance, however, is the factthat the adsorbents containing the polymeric spacers are inherently muchmore effective in extracting the toxin. It is notable that thesederivatives are quite effective even when diluted 50-fold withunsubstituted agarose. The most preferred derivative appears to beA-NatAlb-gang² ; with this adsorbent some adsorption is detectable evenafter a 600-fold dilution. Furthermore, leakage is not a problem in thiscase since no significant free ganglioside is detectable in effluents ofcolumns containing a 10-fold diluted adsorbent. Experiments of thistype, which are quite useful in comparing the relative effectiveness ofa variety of adsorbents, indicate that fetuin-agarose is quite inferiorto any of the ganglioside-agarose derivatives since virtually noadsorption occurs to adsorbents diluted 1:10 with unsubstituted agarose.

The chromatographic behavior of a sample of purified cholera toxincontaining a tracer quantity of [¹²⁵ I]toxin on a column containingA-Alb-gang is presented in FIG. 2 and Table III of said Cuatrecasas etal, 1973e, supra, at page 4257. Adsorption is prevented by incubatingthe toxin with gangliosides before chromatography. In the absence ofgangliosides the column extracts 70% of the protein and more than 95% ofthe lipolytic activity. The protein which does not adsorb to the columnis virtually without lipolytic activity and the radioactivity in thispeak does not bind to liver membranes. Nearly 90% of the lipolyticactivity applied to the column is recovered upon elution with 7 Mguanidine.HCl. These experiments demonstrate that the behavior of ¹²⁵I-labeled and native cholera toxin on such affinity columns is verysimilar.

Chromatography of Crude Vibrio cholerae Filtrates on Affinity Columns.The total material obtained from 3.4 l. of crude culture medium of V.cholerae was chromatographed on a 5-ml column of A-NatAlb-gang (FIG. 3and Table IV of said Cuatrecasas et al, 1973e, supra, at pages4257-4258). More than 99% of the lipolytic activity and 80% [orvirutally all of the active form] of the tracer ¹²⁵ I-labeled toxinpresent in this material disappeared after passage through this column,and no free gangliosides could be detected in the effluent samples.After very thorough and prolonged washing, elution with 5 Mguanidine.HCl resulted in the recovery of about 1 mg of protein and atleast 70% of the ¹²⁵ I-labeled toxin which had adsorbed to the column.On the basis of radioactivity, the toxin was purified more than 90-foldby this procedure.

The material eluted from this column was virtually devoid of biologicalactivity. The lack of activity in this material is not explained by thepresence of gangliosides or of residual guanidine [which inhibitslipolysis] since it did not alter the lipolytic response of nativecholera toxin when these were incubated together before assay. The lackof activity is similarly not explained by the presence of biologicallyinactive choleragenoid, which can block the binding and activity ofcholera toxin [Cuatrecasas, Biochemistry, 12, 3577 (1973d)], sincepreincubation of cells with the eluted material did not block thebinding of ¹²⁵ I-labeled toxin or the lipolytic response to nativetoxin. Data presented suggests that the loss of activity may haveresulted from dissociation of the toxin into subunits, as aforesaid;this process is essentially irreversible when the concentration ofcholera toxin is very low. In some experiments it has been possible toelute about 10-20% of the lipolytic activity adsorbed, although thereasons for such recovery in certain experiments is not known.

In the experiment described in the noted Cuatrecasas et al, 1973e, FIG.3, supra, it was estimated on the basis of lipolytic activity that theentire material applied to the column contained about 3.5 mg of choleratoxin. Since all of the activity and all of the active radioactivitywere removed by the column, and since the recovery of adsorbedradioactivity upon elution was about 70%, it was anticipated thatelution should have yielded about 2.4 mg of protein had the purificationbeen complete. However, only 1.3 mg of protein was present in the elutedsample. The reason for the slight but significant disparity between thequantity of protein actually eluted and that anticipated is notapparent. It is possible that alterations of the native toxin, notreflected in the ¹²⁵ I-labeled material, occur during the step ofconcentration of the crude toxin since this exposes the protein to highionic strength.

Since in the experiment depicted all of the cholera toxin applied on thecolumn was extracted from the sample, the binding capacity of suchcolumns was examined. When the quantity of sample applied was increasedby 2.5 times and the adsorbent was diluted 5-fold with unsubstitutedagarose, only one-third of the cholera toxin applied was adsorbed to thecolumn [FIG. 4, Cuatrecasas et al, 1973e, supra, at page 4259]. Thetoxin was effectively extracted from the first effluent fractions whilevirtually no extraction occurred in the last fractions. As in the otherexperiments, only a very small proportion of the total protein wasadsorbed to the column, and elution of the radioactively labeled toxinwas satisfactory. There was excellent correspondence between theappearance of radioactivity and lipolytic activity in the breakthroughfractions, pointing again to the similarity in the behavior of thelabeled and native toxins. The use of ¹²⁵ I-labeled tracers in theseexperiments greatly facilitates monitoring and quantitation of thechromatographic experiments.

Reversible Denaturation of Cholera Toxin. The binding of cholera toxinto the affinity columns is so strong that to achieve elution it isnecessary to use buffers which are likely to unfold, and possiblydenature, the protein. Because of this, and because the protein elutedfrom columns on which crude samples were chromatographed yieldedessentially inactive toxin preparations, the ability of cholera toxin torenature after removal of denaturants was examined [Table V, Cuatrecasaset al, 1973e, supra, at page 4259]. Brief exposure of [¹²⁵ I]choleratoxin to acidic and basic conditions, and to relatively lowconcentrations of urea and guanidine.HCl, diminishes profoundly theability of the iodoprotein to bind to liver membranes upon dilution orneutralization of the denaturant; similar effects are observed if ureaand guanidine.HCl are removed by dialysis. The ¹²⁵ I-labeled toxin whichis eluted from affinity columns such as those depicted in the aforesaidnoted FIG. 1 and Tables I and II does not bind at all to liver membranesif tested after removal of guanidine.HCl. These results suggest that the¹²⁵ I-labeled toxin is undergoing an irreversible unfolding ordenaturation. The conditions which cause this irreversible effect occurwith concentrations of urea and guanidine.HCl which are lower than thosewhich are required to elute the toxin which is adsorbed to aganglioside-agarose column. This suggests that ganglioside bindinggreatly stabilizes the tertiary or quaternary structure of the protein.

The effects described immediately above suggest results contradictory tothose of the experiments described in the noted FIG. 2, whereguanidine.HCl elution of a chromatographed sample of purified toxinyielded active toxin. The possibility was examined that the irreversibledenaturation described is dependent on the concentration of toxin usedin such experiments. Samples of native cholera toxin [0.1-0.5 mg/ml]containing a tracer of ¹²⁵ I-labeled toxin were exposed for 25 min. at24° to (a) 0.1 M phosphate buffer [pH 7.4], (b) distilled water, (c) 0.1N HCl, (d) 7 M guanidine.HCl, and (e) 7 M urea under conditions similarto those described in the Cuatrecasas et al, 1973e, Table V. The sampleswere then diluted fivefold and dialyzed overnight against large volumesof Krebs-Ringer-bicarbonate buffer. Virtually no radioactivity was lostduring the period of dialysis, and the lipolytic activity of all thesamples was equal to that of the sample exposed only to phosphatebuffer. It is clear that at these concentrations of cholera toxin, whichare about 10,000 times higher than those described in said Table V,denaturation of the toxin by exposure of these solvents is readilyreversible.

There is some evidence that the denaturation described above involves aprocess of dissociation of cholera toxin into subunits. As suggestedabove, exposure of high concentrations [0.5 mg/ml] of toxin to 7 Mguanidine.HCl followed by dialysis does not result in the loss of the¹²⁵ I-labeled toxin which is added as a tracer. In contrast, exposure oftracer quantities [10 ng/ml] of ¹²⁵ I-labeled toxin to 7 M guanidine.HClresults in the rapid loss of radioactivity upon dialysis, even when 0.1%albumin is added to the sample to prevent adsorption to the dialysismembrane. Under these conditions 60% of the radioactivity is lost afterdialysis for 2 hours at 24°, and about 85% is lost after dialysis for 24hours at 4°. Further evidence for the dissociation into subunits comesfrom disc gel electrophoretic experiments in 0.5% sodium dodecylsulfate, Cuatrecasas et al, 1973e, supra, at pages 4260-4262. Theseresults are consistent with findings of LoSpalluto and Finkelstein,Biochim. Biophys. Acta, 257, 158 (1972), who described reversibledissociation of cholera toxin into subunits of about 15,000 molecularweight upon exposure to 6 M urea or to a pH 3.6; these experiments wereperformed at concentrations of toxin varying from 2.5 to 4 mg per ml.

Ganglioside-Agarose and the Lipolytic Response to Cholera Toxin. Theganglioside-agarose derivatives described herein are quite effective inremoving ¹²⁵ I-labeled toxin from buffer solutions when the derivatizedbeads are added and incubated in suspension. The derivatives can bediluted with unsubstituted agarose, and adsorption is generally completeafter incubating for 15 min. at 24°. The derivatives are also quitepotent in protecting fat cells against the metabolic effects of thetoxin provided that the beads are added to the cells before the toxin[Table VI, Cuatrecasas et al, 1973e, supra, at page 4260]. Addition ofthe adsorbent 10 min. after addition of the toxin has no effect on thelipolytic response to the toxin. These results are consistent with thenearly irreversible nature of the binding of cholera toxin to cellmembranes [Cuatrecasas, 1973a, b, supra].

Effect of Water-soluble Polymers Containing Gangliosides. Thewater-soluble copolymer of poly(L-lysine) (backbone) and -(DL-alanine)(side branches) which contains covalently linked gangliosideseffectively inhibits the binding of ¹²⁵ I-labeled cholera toxin to livermembranes [Table VII, Cuatrecasas et al, 1973e, supra, at page 4260].This ganglioside polymer is effective in concentrations which in thefinal incubation medium are as low as 0.1 μg/ml.

The ganglioside-containing polymer is also quite effective in blockingthe lipolytic effect of cholera toxin on fat cells [Table VIII,Cuatrecasas et al, 1973e, supra, at page 4260]. However, as with theinsoluble ganglioside derivatives, marked effects are observed only ifthe polymer is added to the cells before the addition of cholera toxin.These soluble derivatives appear to be more potent than the comparableinsoluble agarose derivatives. Nearly complete inhibition of activity isachieved with concentrations of the polymer as low as 0.5 μg/ml.

Disc gel electrophoretic characterization of cholera toxin andcholeragenoid consistent with the invention too is described atCuatrecasas et al, 1973e, supra, at pages 4260-4262. Experimental

Materials. The crude culture filtrate cholera toxin from Vibrio choleraewas lot 002 [Wyeth], provided by the SEATO Cholera Research Program,NIAID. This material was prepared by lyophilization of culture filtrateof V. cholerae strain 569B, grown in Richardson's [Richardson andNoStle, J. Infec. Dis., 121, Suppl. 73 (1970)] TRY medium; 100 g of thismaterial represented the lyophilized filtrate of about 8.45 l. of crudeculture medium. Cholera toxin [lot 1071], purified by the method ofFinkelstein and LoSpalluto, J. Infec. Dis., 121, Suppl., 563 (1970), wasobtained from SEATO Cholera Research Program; it was prepared undercontract for the National Institute of Allergy and Infectious Diseasesby Dr. R. A. Finkelstein, The University of Texas Southwestern MedicalSchool, Dallas, Texas. Choleragenoid was a gift from Dr. Finkelstein.Bovine brain gangliosides [grades II and III] were purchased from Sigma.Procine thyroglobulin was obtained from Miles, fetuin from Calbiochem,bovine albumin [grade A] from Pentex, guanidine.HCl [Ultra Pure] andurea from Schwarz-Mann, Sepharose 4B from Pharmacia,dicyclohexylcarbodiimide and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide from Pierce, andN-hydroxysuccinimide from Aldrich. The multichain copolymer,poly(L-lysyl-DL-alanine), which consists of a polylysyl backbone and hasan alanine to lysine ratio of 15 to 1, was purchased from Miles; themolecular weight of this compound was about 37,500. Poly(L-lysine).HCl[mol wt 160,000] was obtained from Schwarz-Mann.

Procedures. [¹²⁵ I]Cholera toxin [5-20 μCi/μg] was prepared from toxinsamples chromatographed on Sephadex G-75 by procedures described atCuatrecasas, 1973a, supra. About 80% of the radioactive materialprepared in this way bound specifically to liver membranes. Themolecular weight of cholera toxin was assumed to be 84,000 and the A₁^(1%) _(cm) [280 nm] 11.41 [LoSpalluto and Finkelstein, supra]. Isolatedfat cells were prepared from male Sprague-Dawley rats [90-120 g] by themethod of Rodbell, J. Biol. Chem., 241, 140 (1966). Liver membranes wereprepared by homogenization in 0.25 M sucrose followed by differentialcentrifugation [Cuatrecasas, 1972b, supra]. Protein content wasdetermined by the method of Lowrey et al, J. Biol. Chem., 193, 265(1951) using bovine albumin as the standard.

The specific binding of [¹²⁵ I] cholera toxin to liver membranes wasperformed as described previously [Cuatrecasas, 1973a, b, supra]. Livermembranes [20-100 μg of protein] were incubated for 20 min. at 24° inKrebs-Ringer-bicarbonate buffer [pH 7.4], containing 0.1% albumin andthe iodinated toxin [5-10 × 10⁴ cpm]; binding was determined byfiltration on cellulose acetate (EGWP, Millipore Corp.) filters. Forevery determination nonspecific binding was determined by includingcontrol samples in which native toxin [5 μg/ml] was added to themembranes before adding [¹²⁵ I]toxin. The presence of free gangliosidesin column effluents was determined by measuring the ability of thesesamples to block the binding of [¹²⁵ I]cholera toxin to liver membranes[Cuatrecasas, 1973a, b, supra]. The iodoprotein was incubated with thesample for 50 min. at 24° in Krebs-Ringer-bicarbonate buffer [pH 7.4],containing 0.1% albumin before determining specific binding. By thesemethods it is possible to detect less than 50 ng/ml of crude bovinebrain gangliosides [type III, Sigma].

Lipolysis by fat cells was studied by determining the concentration ofglycerol in the medium by the method of Ryley, Biochem. J., 59, 353(1955). The bioassay of cholera toxin was based on the potent lipolyticaction of the toxin on fat cells. Fat cells [2-8 × 10⁵ cells/ml] wereincubated at 37° in Krebs-Ringer-bicarbonate buffer containing 3°albumin; samples [0.1 ml] were analyzed for glycerol at various timeperiods between 90 and 160 min. [Cuatrecasas, Biochemistry, 12, 3567(1973c)]. Since the absolute lipolytic responses varied betweenexperiments, the activity of unknown toxin samples were expressed on thebasis of comparisons with standard curves obtained with native choleratoxin.

Protein was analyzed by electrophoresis in 7.5% polyacrylamide disc gels[7.5 × 0.5 cm] at pH 7.0 in 0.1 M sodium phosphate buffer, both in thepresence and absence of sodium dodecyl sulfate [Weber and Osborn, J.Biol. Chem., 244, 4406 (1969)]. Protein was detected by staining 1-2hours with Coomassic Brilliant Blue [0.25% in methanol water-acetic acid(5:5:1, v/v)], and gels were destained overnight in water-aceticacid-methanol [35:3:2, v/v]. Molecular weights were estimated fromelectrophoretic mobilities of standard proteins [cytochrome c,ovalbumin, serum albumin] in gels containing 0.1% sodium dodecylsulfate.

Preparation of Ganglioside-Agarose Derivatives. Poly(L-lysine) and thebranched, multichain copolymer of L-lysine ("backbone") and DL-alanine("side arms") were coupled to cyanogen bromide activated agarose by themethods of Sica et al, Nature (London), New Biol., 244, 36 (1973a); Sicaet al, J. Biol. Chem., in press (1973b); Cuatrecasas, J. Biol. Chem.,245, 3059 (1970); copending application, Ser. No. 475,305, now U.S. Pat.No. 3,947,352; and application, Ser. No. 475,314, and copendingcontinuation applicaton, Ser. No. 713,108. These polymers were used toincrease the number of potentially modifiable functional groups [α-aminogroups in the copolymer, ε-amino groups on the homopolymer] on theagarose, to place these groups at a considerable distance from theagarose backbone, and to enhance the likelihood of multipoint attachmentof the soluble polymer on the agarose, which would increase thestability of linkage of subsequently substituted ligands. Thederivatives used contained about 1.2 mg of copolymer/ml of agarose andabout 1.4 mg of poly(L-lysine)/ml of agarose. Albumin was also used as amacromolecular spacer for the same reasons described above. Albumin wascoupled to cyanogen bromide activated agarose in the absence [native] orpresence [denatured] of 10 M urea, as described recently [Sica et al,1973a, b, supra]; these derivatives contain 2-3 mg of albumin/ml of gel.3,3'-Diaminodiproplamine, fetuin, and thyroglobulin were coupled toagarose with cyanogen bromide [Cuatrecasas, 1970, supra]; these agarosederivatives contained about 10 μmol. 6 mg, and 8 mg, respectively, ofthe ligand and proteins per ml of packed gel.

Gangliosides were coupled through carboxy groups of the terminal sialicacid residues to amino groups of the derivatized agaroses by utilizing awater-soluble carbodiimide reagent or dicyclohexylcarbodiimide, bypreparing an active N-hydroxysuccinimide ester of the ganglioside, or bypreparing an activated, mixed anhydride of the ganglioside.

Coupling with Carbodiimides. Ther derivatized agarose [containing aminogroups] [25 ml] was washed and suspended in 50 ml of 50% (v/v) aqueousdioxane. Brain gangliosides [type III, Sigma] [50 mg] were added and thesuspension was gently shaken at 24° for 15 min.1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide [100 mg] was added and thesuspension was shaken for 6 hours at 24°, and another 100 mg portion ofthe carbodiimide was added. After shaking for another 12 hours, the gelwas washed with 500 ml of water, 500 ml of 75% (v/v) aqueous methanol,250 ml of 6 M guanidine.HCl, and 500 ml of water. The content ofganglioside, as judged by the recovery of unreacted ganglioside, wasabout 0.5 mg/ml of gel. Coupling was also performed in an organicsolvent by reacting 20 mg of ganglioside and 5 mg ofdicyclohexylcarbodiimide in 10 ml of dioxane for 30 minutes at 15°. Thiswas added to 20 ml of albumin-agarose suspended in dioxane in a totalvolume of 40 ml. After reacting for 15 hours at 24°, the gel was washedwith 500 ml of dioxane, 500 ml of 90% (v/v) methanol, and 250 ml of 6 Mguanidine. HCl.

N-Hydroxysuccinimide Ester. Ganglioside [20 mg] was reacted with 2.5 mgof N-hydroxysuccinimide and 2.5 mg of dicyclohexylcarbodiimde for 30min. at 15° in 10 ml of dioxane. The solution was then added to 20 ml ofalbumin agarose suspended is dioxane [total volume, 40 ml]. Aftershaking for 15 hours at 24°, the gel washed as above described[water-soluble carbodiimide reaction].

Mixed Anhydride. A 100-μl portion of 0.1 M N-methylmorpholine intetrahydrofuran was added to a solution of anhydrous tetrahydrofurancontaining 20 mg of ganglioside. After stirring the solution for 10 min.at 0°, 100 l of 0.1 M isobutyl chloroformate [Vaughan and Osato, J.Amer. Chem. Soc., 74, 676 (1952)] in tetrahydrofuran was added and thereaction was allowed to continue for 20 min. at 0°. The reaction mixturewas added to 20 ml of the amino agarose derivative suspended in dioxane[total volume, 40 ml]. After reacting for 15 hours at 24° the gel waswashed as above described.

Although all of the methods above described resulted in effectiveadsorbents, the most preferred results were consistently obtained withderivatives prepared with the water-soluble carbodimide and with themixed anhydride.

Preparation of Water-Soluble Polymers Containing Gangliosides.Gangliosides were coupled to the branched-chain copolymer of lysine andalanine with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; 20 mg ofpoly(L-lysyl-DL-alanine) was dissolved in 7 ml of water and 5 ml ofmethanol and 40 mg of ganglioside [dissolved in 10 ml of 50% aqueousmethanol] were added. The mixture was stirred at 24° for 15 min. and two40 mg portions of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide wereadded at 6-hour intervals. After stirring for an additional 12 hours,the reaction mixture was dialyzed against 4 l. of H₂ O for 15 hours andlyophilized. The sample was ten chromatographed on a column [2.4 × 70cm] of Sephadex G-75 equilibrated with 25% aqueous methanol containing0.005 M HCl; the flow rate was 15 ml/hr, 4 ml fractions were collectedand the elution was monitored by continuously recording the absorbanceat 256 nm. The peak in the void volume was collected, lyophilized, andrechromatographed on a column [1.6 × 26 cm] of Sephadex G-100equilibrated with 6 M guanidine.HCl [12 ml/hr, 4 ml/fraction]. Thematerial present in the first peak was dialyzed against four changes ofH₂ O (4 l.) for 24 hours and lyophilized. The yield was 22 mg.

Thus, the present invention demonstrates that columns containingganglioside-agarose derivatives can selectively extract cholera toxineven when the toxin is present in concentrations as low as 10⁻¹¹ M. Themost preferred adsorbents are those which contain macromolecular spacers[i.e., poly(amino acid) polymers, albumin] interposed between theagarose backbone and the covalently attached ganglioside. Some of thesederivatives are still effective after 600-fold dilution withunsubstituted agarose. Such macromolecular adsorbents have recentlyproved most useful in the purification of estrogen receptors from theuterus [Sica et al, 1973a, b, supra]. It has been possible to purifyestrogen receptors about 100,000-fold in a single step by using 20-folddiluted poly(L-lysyl-DL-alanine)-agarose derivatives containingcovalently attached estradiol.

Among the specific advantages of the derivatives having "macromolecular"spacers is a high degree of ligand substitution which permits the use ofthe adsorbent in diluted form, and this results in a decrease in thenonspecific protein adsorptive properties of the gel. Furthermore, theligand is separated from the agarose backbone by large distances, whichin the case of the branched amino acid copolymer may be as great as 150A. The high probability that the interposing macromolecule is anchoredto the insoluble polymer by multiple points greatly increased theoverall chemical stability of the attached ligand by stabilizing thebasic cyanogen bromide attachment of the unit to the agarose. Thisenhanced stability may be quite important in very high-affinity systemssuch as the present one in which leakage of even small quantities of theligand may seriously interfere with the specific adsorptive behavior ofthe proteins. These derivatives, particularly those prepared withalbumin, may in addition present a more favorable microenvironment forthe specific ligand-protein interaction. This appears to be the casewith the estradiol-agarose derivatives [Sica et al, 1973b, supra].

The ability of gangliosides to bind extremely tightly to cholera toxin,and thus to prevent the binding and the biological effects of the toxinin various tissues [Cuatrecasas, 1973a, b, supra], evidenced thatgangliosides are useful therapeutic agents for clincial cholera.However, the demonstration that free gangliosides may be incorporatedspontaneously into cell membranes, and that this can ultimately resultin increased binding of the toxin and in enchanced biological effects[Cuatrecasas, 1973b, supra], suggests that the in vivo use of suchagents may be dangerous if not ineffective. For this reason theinsoluble and soluble polymeric ganglioside derivatives described hereinare more rational agents for the therapeutic value of gangliosides inclinical cholera.

While the invention has been shown and described and pointed out withreference to certain preferred embodiments thereof, those skilled in theart will appreciate that various changes, modifications, substitutions,and omissions can be made by those skilled in the art without departmentfrom the spirit of the invention. It is intended, therefore, that theinvention be limited only by the scope of the claims which follow.

What is claimed is:
 1. In a polysaccharide matrix useful as an adsorbentfor affinity chromatography techniques, the improvement which comprisesa ganglioside molecule covalently coupled to the backbone of saidpolysaccharide matrix.
 2. The polysaccharide matrix as defined by claim1, wherein a spacer moiety is interposed between the polysaccharidematrix and the coupled ganglioside.
 3. The polysaccharide matrix asdefined by claim 2, wherein the spacer moiety comprises a polyfunctionalmacromolecular spacer coupled to the backbone of said polysaccharidematrix in multipoint attachment, the said polyfunctional macromoleculebeing selected from the group consisting of (1) poly-L-lysine, and (2)the graft copolymer, poly(L-lysly-DL-alanine), and the said gangliosidebeing covalently coupled to said polyfunctional macromolecule.
 4. Thepolysaccharide matrix as defined by claim 1, further comprising acholera toxin adsorbed thereto.
 5. The polysaccharide matrix as definedby claim 2, further comprising a cholera toxin adsorbed thereto.
 6. Thepolysaccharide matrix as defined by claim 3, further comprising acholera toxin adsorbed thereto.
 7. The polysaccharide matrix as definedby claim 3, wherein the polyfunctional macromolecule ispoly(L-lysyl-DL-alanine), a graft copolymer of poly-L-lysine (backbone)and poly-DL-alanine (side chains).
 8. The polysaccharide matrix asdefined by claim 7, wherein the poly(L-lysyl-DL-alanine) has an averagemolecular weight of about 37,500 and a lysine to alanine ratio of about1:15.
 9. The polysaccharide matrix as defined by claim 3, wherein thepolyfunctional macromolecule is poly-L-lysine.
 10. The polysaccharidematrix as defined by claim 9, wherein the poly-L-lysine has an averagemolecular weight of about 160,000.
 11. The polysaccharide matrix asdefined by claim 3, wherein the polysaccharide is selected from thegroup consisting of cellulose, starch, cross-linked dextran, andagarose.
 12. The polysaccharide matrix as defined by claim 11, whereinthe polysaccharide is agarose.
 13. The polysaccharide matrix as definedby claim 3, wherein the said polysaccharide-polyfunctionalmacromolecular complex is diluted with an unsubstituted polysaccharidegel.
 14. The polysaccharide matrix as defined by claim 13, wherein thesaid dilution is about 200- to 600-fold.
 15. The polysaccharide matrixas defined by claim 3, wherein the ganglioside is bovine brainganglioside.